Extracellular Vesicle Protein Assay With DNA Barcodes for Early HCC Detection

The assay segments the detection process into distinct functional modules: EV isolation using capture beads, surface protein labeling with DNA-barcoded antibodies, bead capture via click chemistry, and DNA barcode amplification/sequencing. Each module performs a specific function, allowing the complex detection task to be broken down into manageable steps that can be optimized independently while maintaining overall sensitivity.

The patent introduces several intermediary elements to bridge the gap between simple sampling and complex detection: DNA barcodes serve as intermediaries between capture antibodies and detection systems, click chemistry moieties (tetrazine and TCO) act as intermediaries for bead-EV attachment, and surface protein markers serve as intermediaries for EV identification. These intermediaries enable highly sensitive detection without requiring direct complex interactions.

The assay incorporates self-service features to reduce operational complexity: the capture beads self-assemble EVs from plasma samples through affinity binding, the click chemistry reaction automatically occurs when tetrazine-functionalized beads contact TCO-labeled EVs, and the DNA barcodes self-amplify through PCR. These self-driven processes minimize manual intervention and simplify sample processing despite the advanced detection capabilities.

The patent utilizes parameter changes to simplify operation: DNA barcodes enable signal amplification through exponential PCR amplification, converting trace EV signals into detectable quantities. The click chemistry reaction provides rapid, specific binding under mild conditions, and the use of established molecular biology techniques (PCR, sequencing) leverages familiar parameters and protocols for researchers.

The assay performs preliminary enrichment of EVs from plasma samples using affinity capture beads before detection. This preliminary concentration step ensures that even small plasma volumes contain sufficient EVs for sensitive detection. The capture beads selectively bind EVs expressing surface markers, concentrating them from the plasma matrix and enabling downstream analysis with minimal sample input.

The patent replaces mechanical concentration methods (such as ultracentrifugation or filtration) with biochemical affinity-based enrichment. The DNA-barcoded capture antibodies specifically bind to surface proteins on EVs, providing highly selective enrichment that is more efficient and gentler than mechanical methods. This substitution allows sensitive detection from small plasma volumes without the losses associated with mechanical processing.